Protective immune response mediated by neutrophils in experimental visceral leishmaniasis is enhanced by IL-32γ.

Neutrophils are important cells in protection against microbial infections including visceral leishmaniasis (VL). It is well known that IL-32γ increases the protective T helper 17 cell mediated immune response against Leishmania infantum. Thus, in this study we evaluated whether IL-32 γ can increase the protective role of neutrophils against VL. In comparison with wild type (WT) mice, transgenic mice for human IL-32 γ (IL-32 γ Tg) presented a higher frequency and absolute number of neutrophils in both spleen and liver after the establishment of L. infantum infection. The IL-32 concentrations correlated with neutrophil numbers in the infected tissues. The IL-32 γ -induced recruitment of neutrophils was dependent on IL-17, since inhibition of Th17 T cells generation and IL-17 production with digoxin treatment reversed the effects of IL-32 γ. In murine neutrophils, the presence of IL-32 γ enhanced the phagocytosis of L. infantum via CR3. In addition, murine IL-32 γ Tg neutrophils were able to kill L. infantum due to the increased production of ROS when compared with WT neutrophils. In fact, IL-32 γ Tg mice lost their ability to control infection by L. infantum when neutrophils were depleted. In parallel, treatment of human neutrophils with recombinant IL-32 γ increased phagocytosis and ROS-dependent killing of L. infantum, similarly to murine IL-32 γ Tg neutrophils. The data show that IL-32 γ induces neutrophil recruitment to organs affected by VL and increases phagocytosis and killing of L. infantum by neutrophils. Together, data indicate the pivotal axis IL-32 γ -Th17-neutrophils to control VL.

The role of IL-32 in Bacillus Calmette-Guérin (BCG)-induced trained immunity in infections caused by different Leishmania spp.

BACKGROUND: Cells of the innate immune system undergo long-term functional reprogramming in response to Bacillus Calmette-Guérin (BCG) exposure via a process called trained immunity, conferring nonspecific protection to unrelated infections. Here, we investigate whether BCG-induced trained immunity is able to protect against infections caused by different Leishmania spp., protozoa that cause cutaneous and mucosal or visceral lesions. METHODS: We used training models of human monocytes with BCG and subsequent infection by L. braziliensis, L. amazonensis and L. infantum, and the vaccination of wild-type and transgenic mice for IL-32γ before in vivo challenge with parasites. RESULTS: We demonstrated that monocytes trained with BCG presented enhanced ability to kill L. braziliensis, L. amazonensis and L. infantum through increased production of reactive oxygen species. Interleukin (IL)-32 appears to play an essential role in the development of trained immunity. Indeed, BCG exposure induced IL-32 production in human primary monocytes, both mRNA and protein. We have used a human IL-32γ transgenic mouse model (IL-32γTg) to study the effect of BCG vaccination in different Leishmania infection models. BCG vaccination decreased lesion size and parasite load in infections caused by L. braziliensis and reduced the spread of L. amazonensis to other organs in both infected wild-type (WT) and IL-32γTg mice. In addition, BCG reduced the parasite load in the spleen, liver and bone marrow of both WT and IL-32γTg mice infected with L. infantum. BCG vaccination increased inflammatory infiltrate in infected tissues caused by different Leishmania spp. In all infections, the presence of IL-32γ was not mandatory, but it increased the protective and inflammatory effects of BCG-induced training. CONCLUSIONS: BCG's ability to train innate immune cells, providing protection against leishmaniasis, as well as the participation of IL-32γ in this process, pave the way for new treatment strategies for this neglected infectious disease.

Human Interleukin-32γ Plays a Protective Role in an Experimental Model of Visceral Leishmaniasis in Mice.

Visceral leishmaniasis (VL) is a chronic parasitic disease caused by Leishmania infantum in the Americas. During VL, several proinflammatory cytokines are produced in spleen, liver, and bone marrow. However, the role of interleukin-32 (IL-32) has not been explored in this disease. IL-32 can induce production of proinflammatory cytokines in innate immune cells and polarize the adaptive immune response. Herein, we discovered that L. infantum antigens induced expression of mRNA mainly for the IL-32γ isoform but also induced low levels of the IL-32ß transcript in human peripheral blood mononuclear cells. Furthermore, infection of human IL-32γ transgenic mice (IL-32γTg mice) with L. infantum promastigote forms increased IL-32γ expression in the spleen and liver. Interestingly, IL-32γTg mice harbored less parasitism in the spleen and liver than wild-type (WT) mice. In addition, IL-32γTg mice showed increased granuloma formation in the liver compared to WT mice. The protection against VL was associated with increased production of nitric oxide (NO), interferon gamma (IFN-γ), IL-17A, and tumor necrosis factor alpha by splenic cells restimulated ex vivo with L. infantum antigens. In parallel, there was an increase in the number of Th1 and Th17 T cells in the spleens of IL-32γTg mice infected with L. infantum IL-32γ induction of IFN-γ and IL-17A expression was found to be essential for NO production by splenic cells of infected animals. These data indicate that IL-32γ potentiates the Th1/Th17 immune response during experimental VL, thus contributing to the control of L. infantum infection.

Multilevel assessment of carbamazepine effects: An integrative approach using zebrafish early-life stages.

Carbamazepine (CBZ) is the most commonly used drug in epilepsy treatment, and its metabolites are commonly detected among persistent pharmaceuticals in the aquatic environment. This study aimed to investigate CBZ effects on early-life-stage zebrafish (Danio rerio) (from 2 to 168 hpf) by employing of an integrative approach linking endpoints from molecular to individual level: (i) development; (ii) locomotor activity; (iii) biochemical markers (lactate dehydrogenase, glutathione-S-transferase, acetylcholinesterase and catalase) and (iv) transcriptome analysis using microarray. A 168 h - LC50 of 73.4 mg L-1 and a 72 h - EC50 of 66.8 mg L-1 for hatching were calculated while developmental effects (oedemas and tail deformities) were observed at CBZ concentrations above 37.3 mg L-1. At the biochemical level, AChE activity proved to be the most sensitive parameter, as evidenced by its decrease across all concentrations tested (∼25% maximum reduction, LOEC (lowest observed effect concentration) < 0.6 µg L-1). Locomotor behaviour seemed to be depressed by CBZ although this effect was only evident at the highest concentration tested (50 mg L-1). Molecular analysis revealed a dose-dependent effect of CBZ on gene expression. Although only 25 genes were deregulated in organisms exposed to CBZ when compared to controls, both 0.6 and 2812 µg L-1 treatments impaired gene expression related to development (e.g. crygmxl1, org, klf2a, otos, stx16 and tob2) and the nervous system (e.g. Rtn3, Gdf10, Rtn3), while activated genes were associated with behavioural response (e.g. prlbr and taar). Altogether, our results indicate that environmentally relevant CBZ concentrations might affect biochemical and genetic traits of fish. Thus, the environmental risk of CBZ cannot be neglected, especially in a realistic scenario of constant input of domestic effluents into aquatic systems.

Interleukin-32γ in the Control of Acute Experimental Chagas Disease.

Chagas disease (CD) is an important parasitic disease caused by Trypanosoma cruzi. Interleukin-32 (IL-32) plays an important role in inflammation and in the development of Th1/Th17 acquired immune responses. We evaluated the influence of IL-32γ on the immune response profile, pathogenesis of myocarditis in acute experimental CD, and control of the disease. For this, C57BL/6 wild-type (WT) and IL-32γTg mice were infected subcutaneously with 1,000 forms of Colombian strain of T. cruzi. In the histopathological analyzes, T. cruzi nests, myocarditis, and collagen were quantified in cardiac tissue. Cytokine productions (IL-32, IFN-γ, TNF-α, IL-10, and IL-17) were measured in cardiac homogenate by ELISA. The IL-32γTg mice showed a better control of parasitemia and T. cruzi nests in the heart than WT mice. Infected-WT and -IL-32γTg mice showed similar levels of IFN-γ, TNF-α, and IL-17, but IL-10 was significantly higher expressed in IL-32γTg than in WT mice. The cytokine profile found in IL-32γTg animals contributed to body weight maintenance, parasitemia control, and survival. Our results indicate that the presence of human IL-32γ in mice infected with the Colombian strain of T. cruzi is important for infection control during the acute phase of Chagas disease.

Molecular mechanisms of the defective hepcidin inhibition in TMPRSS6 mutations associated with iron-refractory iron deficiency anemia.

Matriptase-2 is a transmembrane serine protease that negatively regulates hepcidin expression by cleaving membrane-bound hemojuvelin. Matriptase-2 has a complex ectodomain, including a C-terminal serine protease domain and its activation requires an autocatalytic cleavage. Matriptase-2 mutations have been reported in several patients with iron-refractory iron deficiency anemia. Here we describe a patient with 2 missense mutations in the second class A low-density lipoprotein receptor (LDLRA) domain. Functional studies of these 2 mutations and of a previously reported mutation in the second C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1 (CUB) domain were performed. Transfection of mutant cDNAs showed that membrane targeting of the 2 LDLRA mutants was impaired, with Golgi retention of the variants. The activating cleavage was absent for the LDLRA mutants and reduced for the CUB mutant. All 3 mutated proteins were still able to physically interact with hemojuvelin but only partially repressed hepcidin expression compared with wild-type matriptase-2. Our results underline the importance of LDLRA and CUB domains of matriptase-2.

New world Leishmania spp. infection in people living with HIV: Concerns about relapses and secondary prophylaxis.

Coinfection with the human immunodeficiency virus (HIV) and Leishmania impairs immune responses, increases treatment failure and relapse rates in patients with American tegumentary leishmaniasis (ATL), as well as visceral leishmaniasis (VL). There is insufficient data on the treatment, relapse, and secondary prophylaxis in patients coinfected with HIV/Leishmania in Brazil. This study investigated patients with HIV/ATL and HIV/VL to describe the outcome of leishmaniasis in patients assisted at a referral hospital of Brazilian midwestern region. Patients with HIV/ATL (n = 21) mainly presented cutaneous diseases (76.2%) with an overall relapse rate of 28.57% after treatment, whereas HIV/VL (n = 28) patients accounted for 17.5% of the cases. The counts of CD4+ T cells and CD8+ T cells and the CD4+/CD8+ cell ratios at diagnosis or relapses were not significantly different between relapsing and non-relapsing patients. Patients with HIV/ATL or HIV/VL showed high levels of activation markers in CD4+ and CD8+ T cells. The regular use of highly active antiretroviral therapy (HAART) and viral load at the time of diagnosis did not influence the relapse rates. Relapses occurred in 36.4% (4/11) of the patients with HIV/VL receiving secondary prophylaxis and in 5.9% (1/17) of the patients who did not receive secondary prophylaxis (p = 0.06). These data are relevant for the therapeutic management of the patients coinfected with HIV/Leishmania.

IL-15 enhances the capacity of primary human macrophages to control Leishmania braziliensis infection by IL-32/vitamin D dependent and independent pathways.

How human macrophages can control the intracellular infection with Leishmania is not completely understood. IL-15 and IL-32 are cytokines produced by monocytes/macrophages that can induce antimicrobial mechanisms. Here, we evaluated the effects of recombinant human IL-15 (rhIL-15) on primary human macrophage infection and response to L. braziliensis. Priming with rhIL-15 reduced the phagocytosis of L. braziliensis and increased the killing of the parasites in monocyte-derived macrophages from healthy donors. rhIL-15 induced TNFα and IL-32 in uninfected cells. After infection, the high levels of rhIL-15-induced TNFα and IL-32 were maintained. In addition, there was an increase of NO and an inhibition of the parasite-induced IL-10 production. Inhibition of NO reversed the leishmanicidal effects of rhIL-15. Although rhIL-15 did not increase L. braziliensis-induced reactive oxygen intermediates (ROS) production, inhibition of ROS reversed the control of infection induced by rhIL-15. Treatment of the cells with rhIL-32γ increased microbicidal capacity of macrophages in the presence of high levels of vitamin D (25D3), but not in low concentrations of this vitamin. rhIL-15 together with rhIL-32 lead to the highest control of the L. braziliensis infection in high concentrations of vitamin D. In this condition, NO and ROS mediated rhIL-32γ effects on microbicidal activity. The data showed that priming of human macrophages with rhIL-15 or rhIL-32γ results in the control of L. braziliensis infection through induction of NO and ROS. In addition, rhIL-32γ appears to synergize with rhIL-15 for the control of L. braziliensis infection in a vitamin D-dependent manner.

Genetic variation in Interleukin-32 influence the immune response against New World Leishmania species and susceptibility to American Tegumentary Leishmaniasis.

Interleukin-32 is a novel inflammatory mediator that has been described to be important in the immunopathogenesis and control of infections caused by Leishmania parasites. By performing experiments with primary human cells in vitro, we demonstrate that the expression of IL-32 isoforms is dependent on the time exposed to L. amazonensis and L. braziliensis antigens. Moreover, for the first time we show the functional consequences of three different genetic variations in the IL32 (rs4786370, rs4349147, rs1555001) modulating IL-32γ expression, influencing innate and adaptive cytokine production after Leishmania exposure. Using a Brazilian cohort of 107 American Tegumentary Leishmaniasis patients and a control cohort of 245 healthy individuals, the IL32 rs4786370 genetic variant was associated with protection against ATL, whereas the IL32 rs4349147 was associated with susceptibility to the development of localized cutaneous and mucosal leishmaniasis. These novel insights may help improve therapeutic strategies and lead to benefits for patients suffering from Leishmania infections.

Exposure to dilute concentrations of bupropion affects zebrafish early life stages.

Psychiatric pharmaceuticals are one of the most prescribed active substances globally. Bupropion (BPP) is an antidepressant that acts via inhibition of norepinephrine and dopamine reuptake. It has been found in various water matrices, and thus its effects on aquatic organisms must be studied. The present study aimed to evaluate the acute toxic effects of BPP on zebrafish (Danio rerio) early life stages. For developmental analysis, organisms were exposed for 168 h to concentrations ranging from 0 to 82000 µg/L. Two other experiments were performed by exposing embryos to a wide range of concentrations (from 0 to 50000 µg/L) in order to evaluate BPP effects on embryonic behavior, using the Zebrabox and testing at the biochemical level (acetylcholinesterase, glutathione-S-transferase, lactate dehydrogenase and catalase). Developmental analysis indicated that BPP had low acute toxicity with a calculated 168 h-LC50 of 50346 µg/L. Concentrations equal to or above 44800 µg/L elicited several effects such as hatching delay, edemas and tail deformities. However, concentrations from 7300 µg/L upwards elicited equilibrium alteration. Behavioral analysis showed that BPP affected zebrafish locomotor behavior by decreasing activity at 0.6 µg/L, increasing activity at 8.8 and 158 µg/L, and decreasing activity at 50000 µg/L. Biochemical analysis showed an increase of AChE activity at 158 and 2812 µg/L, an increase in GST at the highest concentrations, CAT alteration and increase of LDH at 0.6, 2812 and 50000 µg/L. We can conclude that BPP affects zebrafish early life stages at environmental concentrations.

ß-Glucan-Induced Trained Immunity Protects against Leishmania braziliensis Infection: a Crucial Role for IL-32.

American tegumentary leishmaniasis is a vector-borne parasitic disease caused by Leishmania protozoans. Innate immune cells undergo long-term functional reprogramming in response to infection or Bacillus Calmette-Guérin (BCG) vaccination via a process called trained immunity, conferring non-specific protection from secondary infections. Here, we demonstrate that monocytes trained with the fungal cell wall component ß-glucan confer enhanced protection against infections caused by Leishmania braziliensis through the enhanced production of proinflammatory cytokines. Mechanistically, this augmented immunological response is dependent on increased expression of interleukin 32 (IL-32). Studies performed using a humanized IL-32 transgenic mouse highlight the clinical implications of these findings in vivo. This study represents a definitive characterization of the role of IL-32γ in the trained phenotype induced by ß-glucan or BCG, the results of which improve our understanding of the molecular mechanisms governing trained immunity and Leishmania infection control.

Exposure to ayahuasca induces developmental and behavioral alterations on early life stages of zebrafish.

Ayahuasca is a psychoactive concoction prepared from the plants Banisteriopsis caapi and Psychotria viridis which are used ancestrally by Amazonian Indian populations and more recently, by Christian religious groups in Brazil and other countries. The aims of the present study were to identify the effects of ayahuasca on zebrafish embryo development and neurobehavior. Toxicity and developmental endpoints for zebrafish embryos were assessed from 0 to 1000 mg/L over 96 h of exposure. The effects on locomotor activity of zebrafish larvae were assessed using a video tracking system (ZebraBox) from 0 to 20 mg/L and after 120 and 144 h of exposure. The LC50 of ayahuasca in zebrafish was determined as 236.3 mg/L. Ayahuasca exposure caused significant developmental anomalies in zebrafish embryos, mainly at the highest concentration tested, including hatching delay, loss of equilibrium, edema and the accumulation of red blood cells. Embryo behavior was also significantly affected, with decreased locomotor activity at the highest concentration tested. These results are in accordance with data obtained in mammal studies highlighting the possible risks of uncontrolled use of ayahuasca. Further research employing more specific behavior analysis could provide additional data on both therapeutic benefits and possible toxicological risk of ayahuasca.

IL-32γ promotes the healing of murine cutaneous lesions caused by Leishmania braziliensis infection in contrast to Leishmania amazonensis.

BACKGROUND: Interleukin 32 (IL-32) is a pro-inflammatory cytokine induced in patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. Here, we investigated whether IL-32 is also expressed in patient lesions caused by L. amazonensis. In addition, we evaluated experimental L. amazonensis and L. braziliensis infections in C57BL/6 transgenic mice for human IL-32γ (IL-32γTg) in comparison with wild-type (WT) mice that do not express the IL-32 gene. RESULTS: Human cutaneous lesions caused by L. amazonensis express higher levels of IL-32 than healthy control skin. In mice, the presence of IL-32γ promoted the control of cutaneous lesions caused by L. braziliensis, but not lesions caused by L. amazonensis in an ear dermis infection model. In addition, IL-32γTg mice displayed less tissue parasitism and inflammation in IL-32γTg than WT mice during the healing phase of L. braziliensis infection. Production of antigen-specific pro-inflammatory cytokines was higher in IL-32γTg mice than in WT mice during L. braziliensis infection but not during L. amazonensis infection. CONCLUSIONS: Human cutaneous lesions caused by L. amazonensis express high levels of IL-32. In mice, the presence of IL-32γ contributes to the lesion healing caused by L. braziliensis but not by L. amazonensis. Data suggest that despite the ability for both species to induce IL-32 in humans, the connections between this cytokine and other immune players induced by related species of parasites can lead to distinct outcomes of the murine infections.

Incidência de constipação intestinal em estudantes de nível superior do sexo feminino / Incidence of intestinal constipation in female college students

Esta pesquisa teve como objetivo avaliar a incidência e as características da constipação intestinal em estudantes universitárias. Foi desenvolvida uma pesquisa descritiva com 104 estudantes do sexo feminino do Curso de Fisioterapia da UNIFRA, Santa Maria/RS.Os instrumentos utilizados foram o questionário de Roma III; uma ficha de avaliação e a escala de Bristol, respondidos pelas acadêmicas do curso de Fisioterapia. A incidência de constipação intestinal foide 59,62% para os critérios de Roma III. Observou-se relevância nas questões referentes ao esforço evacuatório, sensação de esvaziamento incompleto e utilização de manobras manuais, questões específicado questionário de Roma III. A sintomatologia mais referida foi a flatulência abdominal, com 49,04% da amostra. Se não tratada, a constipação pode desencadear patologias pélvicas como prolapsos e incontinências. Por isso, sugere-se a implantação do autocuidado para os futuros profissionais dos cursos de fisioterapia.

This research aimed to evaluate the incidence and the characteristics of intestinal constipation in college students. A descriptive research was developed with 104 female students of the Physical Therapy Course at UNIFRA, Santa Maria/RS. The instruments used for this research were the Rome III questionnaire, an evaluation form, and the Bristol scale, answered by the Physical Therapy students. The incidence of intestinal constipation was of 59.62% for theRome III criteria. It was observed relevance in the questions referring to the defecation effort, sensation of incomplete emptying and use of manual maneuvers, which are specific questions of the Rome III questionnaire. The most reported symptoms were abdominal flatulence, with 49.04% of the sample. If not treated, constipationmay trigger pelvic pathologies as prolapses and incontinences. For this, it is suggested the implantation of self caring for the future professionals of Physical Therapy courses.